"Cooper was a marvel – warts and all was his USP, an almost clinical portrayal of Homo sapiens, the great topographer of the human face, and yet to many people the name means nothing," Mould said.

Cooper was regarded as the best portrait painter of his day, but only worked in miniatures. The Cromwell is best known from a full-size version painted by Peter Lely, where the wart appears as a mere smudge over the Lord Protector's right eyebrow. The face in Cooper's original, in watercolour on vellum, is the size of a 50p piece but miraculously detailed – from the bald patch, creased forehead and roughened cheeks to the jowly five o'clock shadow.

Cooper had painted an earlier portrait of Cromwell soon after he came to power, in which he appears almost Botoxed, the face longer and more conventionally noble. When Cromwell came to his London studio – Cooper demanded that his clients, even royalty, come for an extraordinary eight sittings – he gave the famous order for less flattery and more accuracy.

"It is the best painted wart in English art, if not the only painted wart in English art," Bendor Grosvenor, head of research at the gallery, said. "When you see it close up in high definition the top is all white and flaky, absolutely repulsive."

When Cromwell died his son came to the studio to buy the sketch: Cooper's usual price was £20, but he demanded and got £100. It stayed in Cromwell's family until the 19th century when it was bought by the Duke of Buccleuch, whose descendant has loaned it to the exhibition. Other loans have come from aristocratic private collections, museums, and the Queen.

During Cromwell's reign Cooper painted both leading royalists and parliamentarians, and as soon as Charles II came to power, he too headed for the studio – the artist kept the magnificent sketch from life, like the Cromwell, so he could turn out copies on demand.

Mould and Grosvenor both have form as art detectives, regularly turning up works believed long since lost or destroyed. This time they have identified a youthful man in a very good brown coat, from the V&A collection, as a Cooper self-portrait, and a deceptively meek woman in a plain white cap and gown from a country-house collection – wrongly identified on the 19th century gold frame as a Restoration beauty, Mrs Middleton – as the king's outrageous mistress Nell Gwynn. Her gown is a nursing jacket, suggesting it was painted just after she had a son by Charles. She is said to have called "come here, you little bastard" to the boy in front of the horrified king. When Nell asked what else she could call him, the King promptly made the toddler Duke of St Albans.

In 1672 a contemporary diarist noted the death of Cooper and called him "the most famous Limner of the World". Now, outside art experts, his name is hardly remembered. Grosvenor believes the problem is the nature of miniatures: fragile, hard to display, almost impossible even with modern technology to photograph. "When you see the miniatures in museums they're almost always covered with a cloth which you're invited to lift – but nobody does," Grovenor said. "It would be nice if this time people remember his name, not just the wart."